Method, kit and system for imaging a blood sample

ABSTRACT

Provided is a method for imaging a blood sample and a kit and system for executing the method. The method includes introducing a cell suspension including red blood cells onto a base surface of a carrier having a vertical height (H) being greater than or equal to a vertical depth (h) of the cell suspension when on the base carrier, the cell suspension including a cell concentration (C) being determined by a defined function; allowing the cells in the cell suspension to settle on the base surface of the carrier to form a monolayer of cells thereon; and acquiring at least one microscope image of at least a portion of the monolayer of cells; wherein the at least one microscope image is obtained by a microscope set to Depth Of Field that is not more than 20% of the vertical height of the cell suspension settled on the base surface.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S . Ser. No. 14/440,864to Pollak (published as US 2015/0316477), which is the US national phaseapplication of PCT Application No. PCT/IL/2014/050585 to Pollak(published as WO 15/001553), filed Jun. 30, 2014, which claims priorityfrom Israel Patent Application No. 227276, filed Jul. 01, 2013.

TECHNOLOGICAL FIELD

The present disclosure is in the field of microbiology and in particularto methods relating to cell sample preparations and imaging thereof, foruse, inter alia, in diagnosis.

PRIOR ART

References considered to be relevant as background to the presentlydisclosed subject matter are listed below:

-   -   Anthony Moody Various rapid diagnostic tests for Malaria        parasite in Clinical Microbiology Reviews January 2002 p. 66-78;    -   Vink J P. et al. An automatic vision-based malaria diagnosis        system Journal of Microscopy, 2013, p. 1-13; International        patent application publication No. WO 2010/116341;    -   “Counting blood cells with countess Automated Cell Counter”        found at        http://www.lifetechnologies.com/content/dam/LifeTech/migraction/files/cell-tissue-analysis/pdfs.par83996.file.dat/w-082149-countess-application-blood-cells.pdf;    -   U.S. Pat. No. 4,209,548;    -   U.S. Pat. No. 4,494,479;    -   U.S. Pat. No. 6,819,408;    -   Leif R C. et al Methods for Preparing Sorted Cells as Monolayer        Specimens, Springer Lab Manuals 2000 p. 592-619;    -   Zahniser D J et al. Automated Slide Preparation System for the        Clinical Laboratory, Cytometry 1996 Mar. 15; 26(10):60-4;    -   Knessel E A et al. Roche Image Analysis Systems, Inc. Acta        Cytologica 1996; 40:60-66;

Acknowledgement of the above references herein is not to be inferred asmeaning that these are in any way relevant to the patentability of thepresently disclosed subject matter.

BACKGROUND

Cell slides are ordinarily prepared during a cytopathology procedure,i.e. studying and diagnosing diseases on the cellular level. One of themost prevalent methods of slide preparation is smearing. The samples aresmeared across a glass microscope slide for subsequent staining andmicroscopic examination. The smearing method is required in order toacquire a thin layer of cells on the slide, thus enabling focusing onand imaging the cells. However, smearing might cause a change in cellmorphology. In addition, with smearing it is difficult to accurately andstably image living cells, for at least the reason that cells dry outquickly and additional staining without fixation is almost impossible.

Anthony Moody describes [Various rapid diagnostic tests for Malariaparasite in Clinical Microbiology Reviews January 2002 p. 66-78] interalia, preparation of thin blood films containing a monolayer of redblood cells and multilayered thick blood films.

Reference to Moody is later made by Vink J P. et al. [An automaticvision-based malaria diagnosis system Journal of Microscopy, 2013, p.1-13] which describes a quantitative cartridge-scanner system forvision-based malaria diagnosis, focusing on low malaria parasitedensities. The proposed cartridge allows the formation of a thin bloodfilm and detection of Plasmodium falciparum. To be able to determine theparasite density, Vink et al. aimed at forming a thin blood filmcontaining a monolayer of red blood cell and based their design on thecartridge described in International patent application publication No.WO 2010/116341 (US patent application publication No. 20120225446).

Specifically, WO 2010/116341 describes an apparatus for producing thinlayers of a fluid sample for analysis, that has a two dimensional arrayof analysis chambers, and a branching pattern of entry channels coupledto the array to enable the analysis chambers to be filled in parallel.The analysis chambers are planar with a height less than that of theentry channels so as to produce the thin layers when filled with thefluid sample. The analysis chambers can be suitable for capillaryfilling by a specified fluid sample such as blood. The analysis chambersshould not be more than 15 μm high in order for the cells to form amonolayer. Manufacturing of chambers having height of this order is notalways possible and is relatively expensive. U.S. Pat. No. 4,209,548describes a method wherein a blood sample on a slide is spun to create amonolayer of randomly distributed red blood cells. To inhibit cellmorphology distortion from occurring during drying, the morphologies ofthe cells contained in the monolayer are preserved by a fixing agentafter monolayer preparation but prior to drying. U.S. Pat. No. 4,494,479describes a device for preparing a monolayer film of a biological fluidsample on a slide device that includes a base for retaining a slidethereon and a spreader manually movable linearly relative to the baseand slide in a pass which spreads a sample of the fluid on the slideinto such a monolayer.

The publication “Counting blood cells with countess Automated CellCounter” describes preparation of blood samples for counting white/redblood cells that involves dilution of the blood cells.

U.S. Pat. No. 6,819,408 describes a method and apparatus for analyzing ablood or another biological fluid sample in a quiescent state withoutthe need for additional diluting reagents or fluid streams passingthrough the apparatus during the analytic process. The method andapparatus allow enumeration of particulate constituents of biologicalsamples and inspection thereof using an optical scanning instrument.

Leif R C et al (Methods for Preparing Sorted Cells as MonolayerSpecimens) Springer Lab Manuals 2000 describes the application of amethod of centrifugal cytology for creating a monolayer from cells thatwere previously sorted using a cell sorter (FACS). According to Lief,Centrifugal Cytology is the process where cells in suspension arecentrifuged onto a substrate and then fixed concurrently with theapplication of centrifugal force.

Knessel E A et al (Roche Image Analysis Systems) Acta Cytologica 1996describes the application of ma a batch centrifugation process togetherwith a computer controlled robotic pipetting station to prepared amonolayer from a suspension of cervical sample.

Zahniser D J et al. (Automated Slide Preparation System for ClinicalLaboratory) Cytometry 1996, describes an automated device that collectscells from suspension and disperses them as a monolayer on a glass slideusing filter-transfer technology.

GENERAL DESCRIPTION

The present disclosure provides a method for imaging a blood sample, themethod comprising:

-   -   introducing a cell suspension comprising red blood cells, onto a        base surface of a carrier having a vertical height (H) being        greater than or equal to a vertical depth (h) of said cell        suspension when on said base carrier, the cell suspension        comprising a cell concentration (C) being determined by the        function:        C=F/(h*pi/4*d ²)        -   (F) being a desired base surface coverage; and (d) being an            average cell dimension of the cells in the cell suspension;    -   allowing the cells in the cell suspension to settle on said base        surface of the carrier to form on the base surface of the        carrier a monolayer of cells;    -   acquiring at least one microscope image of at least a portion of        the monolayer of cells,

wherein said at least one microscope image is obtained by a microscopeset to Depth Of Field (DOF) that is not more than 20% of the verticalheight of the cell suspension when settled on said base surface.

Also provided by the present disclosure, a kit for imaging a bloodsample, the kit comprising:

-   -   a carrier comprising a base surface and a vertical height (H);        and    -   instructions for performing the steps of:        -   providing a cell suspension from a blood sample comprising            red blood cells, the cell suspension being of a cell            concentration (C) determined by the function:            C=F/(h*pi/4*d ²)    -   (F) being a desired base surface coverage; and (d) being an        average cell dimension of the cells in the cell suspension;    -   introducing the cell suspension of the desired concentration C        onto the base surface of the carrier, the cell suspension having        said vertical depth (h) when in said carrier, said vertical        depth (h) being smaller or equal to the vertical height (H);    -   allowing the cells in the cell suspension to settle on said base        surface of the carrier to form onto the base surface a monolayer        of cells;    -   acquiring at least one microscope image of at least a portion of        the monolayer of cells,        -   wherein said at least one microscope image is obtained by            setting the microscope to a Depth Of Field (DOF) that is not            more than 20% of the vertical height of the cell suspension            when settled on said base surface.

Yet further, there is provided by the present disclosure a system forimaging a blood sample comprising:

-   -   one or more reservoir units for holding, respectively, one or        more sample treatment agents comprising at least one blood cells        diluting agent;    -   a blood sample preparing unit being in fluid communication with        said one or more reservoir units and configured to receive a        blood sample comprising red blood cells and amount of at least        one blood cell diluting agent and to form therefrom a blood        cells suspension, the amount of said at least one cell diluting        agent being determined so as to dilute said sample of cells by a        dilution factor (D) so as to provide a cell concentration (C);    -   a microscope image acquisition unit for acquiring at least one        image of the blood cells suspension when on a base surface of a        carrier, the carrier having a vertical height (H) being greater        or equal to a vertical depth (h) of said cell suspension when on        said base surface;    -   a control unit being configured to:        -   provide dilution factor D of diluting said sample, factor D            being a function of the desired base surface coverage (F),            the average cell dimension d of cell blood cells, and the            vertical depth h of said suspension of cells that provides a            monolayer of the cells when settled on said base surface of            the carrier; and        -   acquire at least one microscope image of the cell suspension            by a microscope set to a Depth Of Field (DOF) that is not            more than 20% of the vertical height of the cells suspension            when settled on said base surface.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIGS. 1A-1C illustrate components of a system in accordance withnon-limiting embodiments of the present disclosure and a carrier to beused in accordance with some embodiments;

FIG. 2 is a microscope image of Trypanosoma brucei parasites from aperipheral blood sample, captured while Trypanosoma brucei parasites areidentified (marked by arrows) in a monolayer obtained in accordance withan embodiment of the invention.

FIG. 3 is a florescent image of Trypanosoma brucei parasites from aperipheral blood sample, captured while Trypanosoma brucei parasites areidentified (marked by arrows) in a monolayer obtained in accordance withan embodiment of the invention.

FIGS. 4A-4B show images of a blood sample obtained as a bright image ofblood cells at 20× magnification with a monolayer coverage of 75% (FIG.4A) and a corresponding fluorescent image stained with Acridine Orangefluorescent dye (λ=570 nm) at a depth of field of 2.3 μm (FIG. 4B)showing presence of a pathogen as indicated by an arrow.

FIGS. 5A-5B show images of a blood sample obtained as a bright image ofblood cells at 20× magnification with a monolayer coverage of 80% (FIG.5A) and its corresponding florescent image (showing emissions at 460 nm)stained with Hoechst 33342 (FIG. 5B) In the image we show our ability todiagnose malaria causing Plasmodium trophozoites by distinguish betweenthe malaria pathogen and platelets which are much smaller than RBCwithin the sample.

DETAILED DESCRIPTION OF EMBODIMENTS

The present disclosure is based on the understanding that there is aneed in the art of cell preparates for providing cells in a monolayer ina quick (in a scale of seconds or few minutes) and reproducible methodso as to allow quick imaging of small objects in a cell sample, inparticular, a blood sample. To this end, the inventors have developed asimple method that does not necessitate the use of expensive equipment,or to wait until the sample dries before microscope examination.

Specifically, the present invention provides a method comprisingintroducing a cell suspension comprising red blood cells, onto a basesurface of a carrier having a vertical height (H) being greater than orequal to a vertical depth (h) of said cell suspension when on said basecarrier, the cell suspension comprising a cell concentration (C) beingdetermined by the function:C=F/(h*pi/4*d ²)

(F) being a desired base surface coverage; and (d) being an average celldimension of the cells in the cell suspension;

allowing the cells in the cell suspension to settle on said base surfaceof the carrier to form on the base surface of the carrier a monolayer ofcells;

acquiring at least one microscope image of at least a portion of themonolayer of cells,

wherein said at least one microscope image is obtained by a microscopeto Depth Of Field (DOF) that is not more than 20% of the vertical heightof the cell suspension when settled on said base surface.

Optionally, cell concentration C may be calculated using the carrier'svertical height H as an approximation of the sample's vertical depth h,when they are assumed to be approximately equal, such as when filling upthe carrier or a chamber thereof with the sample.

A blood sample may contain a variety of cells, including red bloodcells, platelets and macrophages. As such, in the context of the presentdisclosure, when referring to d it is to be understood as meaning theaverage dimensions of the cells in the sample, taking into considerationthe different dimensions of the variety of cells therein. Since the vastmajority of cells in a blood sample are RBC, d can be taken in someembodiments to be the average dimension of the RBC. The dimension (d) isprovided in mm. The value of d may be determined for example byspectroscopy or from the literature. For example, the average diameterof a human RBC is 6.2-8.2 μm (0.0062-0.0082 mm).

In the above function for determining the cell concentration C, pi isthe constant defining the ratio of a circles circumference to itsdiameters and is roughly equal to 3.14159;

The base surface coverage F as used herein, defines the percent of basesurface area that is covered by cells after settling thereupon, and forthe sake of illustration, when F=1 or near 1, when 100% or almost 100%of the cells are completely covering the base surface. When F=0 thereare no cells on the base surface. In the context of the presentdisclosure, a high F (namely, closer to 1) enables rapid visualizationof a greater number of cells while a low F (namely, closer to 0) enablesa clear distinction between the different cells and in some embodimentsmay also enable more accurate prediction.

In one embodiment, the average base surface coverage is between 40%(F=0.4) and 90% (F=0.9). In some other embodiments, the average basesurface coverage is between 40% and 60% (F being between 0.4 and 0.6).

The vertical depth h may be dictated by a desired minimal or maximaltime interval allotted for sedimentation of the cells on the surfacebase. There is a direct relation between the height (the vertical depth)and time needed for the cells to settle on the base. In someembodiments, for formation of monolayer in a short period of time (e.g.tens of seconds and up to only several minutes), h is in the range of 20μm to 1000 μm. In some other embodiments, h is in the range of 25 μm to600 μm or 30 μm to 250 μm or even 75 μm to 200 μm.

The cells to be images are blood cells (whole blood or RBC sample)comprising at least red blood cells (RBC). In some embodiments, theblood cells are human blood cells. In some embodiments the blood samplecomprises at least one of white blood cells (WBC), bacteria andplatelets.

Further, in some embodiments, the blood sample comprises at least 50%RBC. In some other embodiments, the blood sample comprises at least 75%RBC.

The average concentration of blood cells in a blood sample withdraw froma living entity is known in the art. For example, the highest normal redblood cell (RBC) concentration in blood for women is 4.2 to 5.4million/μl, for men is 4.7 to 6.1 million/μl and for children is 4.6 to4.8 million/μl. When using a concentration known in the literature, itmay be useful to take an average value or a maximum normal value and useit as a base value for dilution so as to obtain the desiredconcentration C. For example, for a human blood sample a value of about6M cells/μl may be used. Alternatively, the cells in the blood samplemay be counted or estimated (manually or automatically, as known in theart) before dilution and/or after to as to have a precise cell count fora given sample.

The desired concentration C of the cell suspension is such that if avolume of the cell suspension is placed on the base surface of a carriersuch that the cell suspension has a determined or estimated verticaldepth, and all or practically all cells are allowed to settle on thesurface of the carrier, a monolayer is formed on the surface with no orlittle overlap between the cells.

The vertical depth h of the cell suspension may be determined or imposedor estimated. For example, when introducing the cell suspension onto abase surface of a carrier having a vertical height H, the maximum valueof h is equal to H. Thus the vertical depth h of the cell suspension maybe assumed to equal the vertical height H of the carrier, when thecarrier is essentially completely filled by the cell suspension. If asmaller amount of the cell suspension is introduced onto the basesurface, the vertical height H will not be completely filled, and hencethe vertical depth h may be calculated or estimated based on the degreeof filling and/or by dividing the volume of the cell suspension by thesurface area which it covers.

Settling of the cells on the base surface of the carrier may take fromseveral seconds to several minutes, for example without applying anexternal force (e.g. mechanical or centrifugation) to affect theprocess. To this end, the method provides a period of time betweenintroducing the cells onto the carrier and acquiring the image to allowthe cells to settle and form the monolayer. In some embodiments, theperiod of time is for not more than 5 minutes, at times, not more than 2minutes, or even not more than 90 seconds, after which a desiredmonolayer is formed on the surface of the carrier's base. In someembodiments, settling involves maintaining the base surface in ahorizontal position, and a time interval of between 20 seconds to 5minutes following introduction of the cell suspension over said basesurface. The time interval being, at times, dictated by the height ofthe sidewalls of the carrier (vertical height (H)) and the verticaldepth h of the suspension (i.e. minutes per mm of h). During this timeinterval the carrier may be maintained essentially motion free. In someembodiments, the time interval until a monolayer is formed is not lessthan 20 seconds, at times, not less than 30 seconds.

In the context of the present disclosure, when referring to “monolayer”of cells it is to be understood as encompassing the distribution ofcells on a surface as an essentially single layer, where at least 50%,at times at least 60%, 70%, 80% or even 90% of the cells are in directcontact with the base surface of the carrier and not more than 20%, attimes no more than 10% or even no more than 5% of the cells overlay eachother (i.e. no more than 20% of cells lie, partially or completely, oneon top of another). Further, when referring to a “monolayer” it is to beunderstood that at least 5%, at times, at least 10% or even at least 20%of the cells touch each other on the safe base surface.

To provide a monolayer of cells, the sample of cells needs to beintroduced onto the carrier at the desired concentration C. At times,the cells to be introduced onto the carrier are already provided withthe concentration C, albeit, at times, the concentration of the cells issuch that requires dilution.

In order to obtain the concentration C of the blood cells in the sample(which typically mostly RBC), the cells sample may be diluted. As such,and in accordance with some embodiments, the method comprises dilutingthe blood sample by a dilution factor (D) to provide a cell suspension.

Factor D may be calculated based on the desired concentration C and theconcentration of the cells before dilution (C₀), as follows:

$D = \frac{C_{0}}{C}$

Since the desired concentration C is may be calculates asC=F/(h*pi/4*d ²).

As such, Factor D may be calculated using the equation:

$D = \frac{C_{0}}{F\text{/}\left( {h*{pi}\text{/}4*d^{2}} \right)}$

The concentration of cells before dilution (C₀) may be based on countingthe cells in the sample. Counting may be performed by any techniqueknown in the art, including, without being limited thereto, a countingchamber (hemocytometer), plating methods, spectrophotometry, flowcytometry, Coulter counter, as well as by image analysis techniques. Attimes, use may be made of information from literature. For example, thehighest normal red blood cell (RBC) concentration in blood for women is4.2 to 5.4 million/μl, for men is 4.7 to 6.1 million/μl and for childrenis 4.6 to 4.8 million/μl. When using a concentration known in theliterature, it may be useful to take an average value or a maximumnormal value and use it as a base value for dilution. For example, for ahuman blood sample a value of about 6M cells/μl may be used. In suchcases, D may be selected to ensure that the vast majority of normalsamples will be within a desired range of F. It is noted that dilutionby dilution factor D may be performed in one or more dilution steps, solong as the total dilution is by factor D (or the final cellconcentration equals C).

As such, in order to obtain a monolayer of blood cells comprising RBC inaccordance with the present disclosure, the following are taken intoconsideration:

For a concentration of blood cells in a μl of a blood sampleC₀=6,000,000 (C₀ in a normal blood sample is 4,000,000 to 6,100,000cells/μl)

For an average diameter of the cells in the blood d=0.0075 mm;

For a carrier with a vertical height H=0.2 mm and a suspension havingvertical depth h=0.2 mm;

For a desired base surface coverage of F=0.5 (50% of the surface iscovered by a monolayer of cells)

The dilution Factor D is calculated using the above equation would thusbe about 100.

Similarly, under the same conditions but with a carrier having avertical height H=0.1 mm and a suspension having vertical depth h=0.1mm, the dilution factor D is calculates using the above equation, andwould thus specifically be about 50.

In some embodiments, the dilution is by a factor D of between about 50to about 300. In some other embodiments, the factor D is between about75 and about 200. In yet some other embodiments, the dilution is byfactor D of about 100.

In yet some other embodiments, the dilution of the blood samplecomprising RBC is to obtain a cell concentration C in the cellsuspension such that after setting on the base surface of the carrierand forming the desired monolayer, the cells' density on the surface isbetween about 10,000 to about 30,000 cells per mm².

Dilution (by factor D) may be performed using any cell diluting agent,such as a buffer known to be used in the art of cell biology which willbe isotonic at the time of sample preparation. Non-limiting examples ofbuffers include Phosphate Buffered Saline (PBS), buffer comprising4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),2-(N-morpholino)ethanesulfonic acid (MES),piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES),3-(N-morpholino)propanesulfonic acid (MOPS),2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES), 2-(Bis(2-hydroxyethyl)amino)acetic acid (Bicine),3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS orEPPS),3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonicacid (TAPS), N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (Tricine),Ethylene-diamine-tetra-acetic acid (EDTA), Sodium Chloride (NaCl),Tris(hydroxymethyl)aminomethane (Tris)

The formation of a monolayer upon or following introduction of the cellsuspension into the carrier may be performed with no additionalintervention (e.g. no centrifugation or smearing). This may be performedsimply by allowing the sample to rest for a short period of time,(namely, from seconds to several minutes) on a flat horizontal surfaceof the carrier.

Once the cells are settled on the base surface of the carrier and adesired monolayer is formed (as a result of the desired cellconcentration upon said introduction), at least one microscope image ofthe cells is acquired. The image is of at least part of the surfacecovered by the cells.

Microscopic imaging techniques are well known in the art and include,inter alia, optical microscopy, fluorescent microscopy, and the like. Insome embodiments, the microscopic imaging is for the purpose ofdiagnosis. Optionally, both one or more florescent images and one ormore brightfield images are taken.

The inventors found that in order to detect objects within a monolayerof blood cells obtained from a cell suspension of the desiredconcentration C (as discussed above) one should select a particularrange of Depth of Field (DOF) to allow clear visualization of the smallobject within the population of blood cells.

DOF is known as the distance between the nearest and farthest objects ina scene that appear acceptably sharp in an image. DOF is mostly aproperty of the microscope's objective lens and the magnification, thelatter being determined by the resolution of interest. For example, foran object being about 1 μm in dimensions (e.g. schizonts or someplatelets), a resolution of at least 0.5 μm would normally be required;similarly, for an object of about 2 μm in dimension, a resolution of atleast 1 μm would normally be required. This also determines themagnification, and a magnification of at least 20× would be used for aresolution of about 0.5 μm, while a magnification of at least 10× wouldbe used for a resolution of about 1 μm. In this connection, it is notedthat a lens is chosen to provide a desired magnification. Lens ischaracterized by a numerical aperture (NA). For example, a lens for 20×magnification may have a numerical aperture (NA) of about 0.4-0.5, whilea lens for 10× magnification may have a significantly smaller NA.

According to Shillaber equation, DOF relates to NA for a givenwavelength of light (λ) and medium refraction index (Ri):

${DOF} = \frac{\lambda\sqrt{{Ri} - ({NA})^{2}}}{({NA})^{2}}$

Below are provides non-limiting examples of DOF for several commerciallyavailable microscope objectives using 500 nm light and air as the medium(Ri=1.00) between microscope objective and object:

Magnification Numerical Aperture (NA) Depth of Field (DOF)  4× 0.10 5010× 0.25 7.7 20× 0.40 2.9 40× 0.65 0.9 60× 0.85 0.36 100×  0.95 0.17

To obtain the DOF of interest, the method may comprise, in accordancewith some embodiments, selecting a microscope objective lens thatprovides said DOF, wherein said lens permits acquiring an image of atleast one object being no more than 3 μm long at any dimension thereof.

In some embodiments, said lens permits acquiring an image of at leastone object having a height with respect to said base surface of no morethan 3 μm.

The inventors have surprisingly found that preparing a monolayer forhigh resolution microscopy does not necessitate placing the blood inspecial carriers as known in the art, which often have a vertical heightH of 15 μm or less. Instead, by providing a diluted cell sample andinserting it onto the base surface of a carrier having a larger verticalheight H a monolayer may be formed, and this monolayer is well dispersedand may be imaged at a very high resolution which is unexpected in viewof the relatively large vertical depth h of the suspension when placedon the base surface.

The inventors have successfully determined that such monolayer of bloodcells once formed having the desired surface coverage of at least 40%cells (F>0.4), it is sufficient for use at a DOF should that is even 20%or less of the vertical height h of the suspension once placed on thebase surface, at times, no more than 15% the vertical height, or evennot more than 10% the said vertical height. To this end, the inventorshave determined that the vertical height h may be within the range of 30μm to 300 μm, as further discussed below.

As such, and as an example only, for an object having a size of about 1μm (e.g. schizonts and some platelets), a resolution of at least 0.5 μmis normally required, for which a magnification of at least 20× isnormally characterized by the numerical aperture (NA) of about 0.4-0.5.A numerical aperture of 0.4-0.5 may provide a DOF of 2.9 μm which is notmore than 20% of a 30-300 μm vertical height h being defined herein forthe suspension when placed on the surface base.

Similarly, and as a further example only, for an object having a size ofabout 2 μm, a resolution of at least 1 μm would normally be required,and a magnification of at least 10× would thus be used. For thismagnification, a lens with NA of about 0.2-0.25 may be used and thiscorrelates with a DOF significantly larger than that of theaforementioned 20× objective, but still, not more than 20% of a 30-300μm vertical height h being defined herein for the cells when settled onthe surface base.

In line with the above and in accordance with some embodiments of theinvention, imaging is performed at a DOF being between about 0.5 μm andabout 10 μm; at times, between about 0.5 μm and 5 μm.

The above conditions for acquiring the microscope image allow for thedetection of small objects being smaller than red blood cells, such asplatelets and pathogens, within a blood sample. In accordance with someembodiments, the microscope set provides a DOF that allows acquiring animage (one or more) of at least one object being no more than 3 μm longat any dimensions thereof.

As noted above, to allow imaging of these smaller objects, a monolayerof the larger cells in the sample is required, and this typicallyrequires diluting a raw blood sample (optionally comprising ananticoagulant or EDTA) before introducing onto the carrier. However, inaddition to dilution, the cells may be treated (e.g. mixed) with one ormore other reagents, such as stains. Staining may be performed before,during or after placing the sample in the carrier. The stain may be anydye, probe or substrate suitable for cell staining, includingfluorescent dyes, and if a plurality of dyes, probes or substrates isadded, some or all of the stains may be a fluorescent dye. In oneembodiment, at least one stain is a fluorescent dye. In some embodimentsat least one dye is included in a diluting agent.

When referring to a stain it is to be understood as encompassing anychemical or biological substance that is capable of staining a componentof a biological cell, to enhance contract and highlight structures ofthe stained object, be it a cell or part of a cell. The stain may have aclass preference or specificity, e.g. may have preference or specificityto staining of nucleic acids and among the nucleic acid, to DNA or RNA,preference or specificity to amino acids, to lipids, carbohydrates etc.

When referring to preference or predominant staining it is to beunderstood that the stain marks (stains) a cellular component in aparticular color or fluorescence that is at least twice, three times,four times or even 10 times greater in its intensity than its stainingintensity to another cellular component at that same color orfluorescence spectrum.

In some embodiments, when referring to preference or predominantstaining it is to be understood that the stain has affinity (molecularattraction) to one cellular component (in the particular color orfluorescence spectrum) that is at least twice, three times, four timesor even 10 times greater in its affinity to another cellular component(at that same color or fluorescence spectrum).

In some further embodiments, when referring to preference or predominantstaining it is to be understood that the staining of the one componentby the stain is stable or has more stability as compared to its stainingof other components. Stability may be understood to mean that thestaining produced by the stain remains substantially consistent for atleast 30 minutes after being brought into contact with the stain, attimes, at least 1 hour, 2 hours or even 5 hours after staining thesample with the stain having preference to the one component.Alternatively, stability may be understood to mean that the stainingproduced by the stain remains substantially consistent during exposureto light (e.g. light used for fluorescence excitation) for at least 0.25seconds, 1 second, or even 15 seconds of exposure.

In this context, it is to be understood that the stain having preferenceto, for example, DNA, may also stain other cellular components but withlower attraction or lower intensity or with a different florescenceresponse (excitation spectrum and/or emission spectrum) such that itallows the greater enhancement of the one component to which the stainhas preference. For example, a stain may predominantly stain DNA,however, under some other conditions the same stain may stain RNA.

In some embodiments, the stains are not cell type specific. In otherwords, the stain may not be specific to a particular pathogen or to aparticular stage of the life cycle of a particular pathogen or to aparticular cell of the host being infected therewith and will stain acell component irrespective if its origin, e.g. a DNA sequence orstructure per se, an RNA sequence or structure per se, protein per se,etc.

There are a variety of stains that may be used in accordance with thepresent disclosure. In some embodiments, the stain is a chromophore orfluorophore.

Stains such as the Giemsa stain (CAS 51811-82-6) are known aschromogenic—their effect is to provide color or opacity to the sampleand are visible, for example, in bright field microscopy.

In some embodiments, the stain provides fluorescent staining of thesample. Fluorescence is visualized by illuminating the sample with an“excitation” spectrum of light, which results in an “emission” at adistinct spectrum of light.

In some embodiments, the stain is a fluorochromatic dye selected fromthe group consisting of Acridine Orange (AO,N,N,N′,N′-Tetramethylacridine-3,6-diamine, green staining for DNA, redstain for RNA), Giemsa stain which is known as a solution of methyleneblue (3,7-bis(Dimethylamino)-phenothiazin-5-ium chloride), eosin (CASNumber 17372-87-1) and Azure B (Trimethylthionine chloride), EthidiumBromide (3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide), Hoechstfamily (C₂₅H₂₆N₆R, with R representing a variety of possiblesubstituents, such as, without being limited thereto, —OH (Hoechst33258); —CH₂CH₃ (Hoechst 33342), —N(CH₃)₂ (Hoechst 34580), —SO₂NH₂(Hoechst S769121)), DAPI (4′,6-diamidino-2-phenylindole), propidiumiodide (2,7-Diamino-9-phenyl-10 (diethylaminopropyl)-phenanthridiumiodide methiodide), SYBR family, YOYO, DRAQ family, SYTOX family, TOTOfamily, crystal violet (Tris(4-(dimethylamino)phenyl)methyliumchloride), any and all molecular beacons, adjacent probes, nucleaseprobes, light up probes, substrate based probes Hematoxylin stains,Safranin (azonium compounds of symmetrical2,8-dimethyl-3,7-diamino-phenazine), acid-Schiff stains, Masson's stain,Prussian blue and any combination thereof.

In one embodiment, more than one stain is used. For example, the samplemay be stained with two or more stains, comprising at least one stainpredominantly staining DNA to thereby provide differential stainingbetween DNA and at least one other cellular component being differentfrom DNA. Alternatively, a single stain may be used, which is excited attwo different wavebands, thus providing two different colors. Forexample, stains such as AO provide different fluorescence spectra fordifferent cellular components. When AO stains DNA at neutral pH, it hasan excitation maximum at 502 nm (cyan) and an emission maximum at 525 nm(green); when it stains RNA at neutral pH, the excitation maximum shiftsto 460 nm (blue) and the emission maximum shifts to 650 nm (red). Assuch, it allows differential staining between DNA and RNA, depending onthe excitation wavelength and conditions of the sample.

When referring to a combination of two or more stains, it is to beappreciated that the two or more stains may be added to the samplesimultaneously or in sequence.

The method disclosed herein provides the detection of a pathogen in theblood sample. Thus, it is not necessary that all cells be infected andthe method is applicable also when only a portion of the cells (even asingle cell) is infected by a pathogen. The pathogen may be anyinfectious microorganism.

In some embodiments, the pathogen is a eukaryotic parasite, and theparasite may be a one cell parasite.

In some embodiments, the parasite is a blood protozoa selected from thegenus consisting of Trypanosoma (causing Chagas disease and Africansleeping sickness); Plasmodium (causing Malaria); Toxoplasma (causingToxoplasmosis); Babesia (causing Babesiosis).

Specifically, when referring to Plasmodium it is to be understood asencompassing at least any member of the group consisting of Plasmodiumfalciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodiumovale (P. ovale), Plasmodium malariae (P. malariae), and Plasmodiumknowlesi (P. knowlesi).

In some embodiments, pathogen is understood to mean a particular stageof the life cycle of a particular pathogen or group thereof. Forexample, the invention disclosed herein can be applied specifically tothe detection of trophozoites, schizonts or gametocytes of Plasmodiumspecies or P. falciparum in particular.

The carrier to which the sample (typically diluted) is introduced isdefined by its base surface and sidewalls. The sidewalls define thecarrier's vertical height (H) which is to be equal or greater than thevertical depth (h) of a cell sample when introduced onto the basesurface of the carrier (i.e. into the carrier). As such, the carrier maybe any unit having an internal void defined by the base surface and thesidewalls and which is biocompatible with biological cells. Wherereferring to biocompatible it should be understood that the cells atleast remain intact, and that optionally viability and/or functionalityare also essentially maintained. The carrier may be provided indifferent forms.

In some embodiments, the carrier is of a kind suitable in diagnosticimaging. Examples of carriers applicable in accordance with the presentdisclosure include microfluidic channels and a well, such as in a multiwell plate.

The carrier to be used with the invention may be of different kinds andmay have different forms, either a commercially available carrier, orone that is specifically designed, as long as it has a vertical heightof no less than about 20 μm and is capable of holding a cell sample witha vertical depth h (the vertical depth being the distance between thetop surface of the sample in the carrier and the base surface of thecarrier) and, not mandatorily, no more than 1000 μm.

The carrier may have an open end, e.g. open top (e.g. a base with sidewalls), with or without a compatible cover, yet it can be in the form ofa closed cavity with a dedicated narrow inlet for introducing the cells(e.g. in the form of a bottle or microfluidic chamber). In someembodiments, the cell suspension is introduced into the chamber bycapillary forces. At times, one or more inner portions of the carriermay be coated or treated, to become hydrophilic and the capillary forcesare increased.

In some embodiments, the carrier is a well, such as those used for cellculturing, and the well may be covered with a compatible cover. Using acover applied on the cell suspension may overcome the capillary effectof the walls of the well on the sample, when the latter is introduced insmall volumes that may lead to a non-even distribution of the sample inthe well.

The top surface (whether fixed or removable) may extend parallel to thebase surface or to only part thereof. Also, it may be applied before orafter the introduction of the cell suspension onto the base surface.

The carrier or at least the base surface of the carrier may includemeans for physical and/or chemical immobilizing the cells beingintroduced Immobilization may be by the use of cell adhesives. Inaddition or alternatively, the base surface may be electrically chargedto attract cells. For example, in the case of RBCs, by treating the basesurface with poly-lysine which has a positive charge, this will attractthe negatively charged RBCs and help in acquiring a more stablemonolayer. Other examples for compounds that may be used forelectrically charging the surface are aminosilane, epoxy, aldehyde andglutaraldehyde.

When a removable cover is used, it may apply forces onto the sample thathas been introduced on the base surface, and by this to expedite thesedimentation process. On the other hand, the cover might cause ruptureof the cells (for example by sheer weight). One way to prevent orminimize the cells damage is by the use of spaces of size compatible tothe average cell's size.

Additionally, the spacers may also act to create a more uniform layerthickness and thus a more uniform distribution of cells on the basesurface of the carrier.

These spaces may be in the form of microparticles or beads, acting aspillars or supporting bodies holding the cover over the cell suspension,without causing any damaging pressure on the cells. Exemplary shapes ofspaces may include, without being limited thereto, spacers having acylindrical shape, with a circular cross section, oval cross section,hexagonal cross section, a star shape cross section. Further, the spacermay be in the form of a disc, or may be in the form of beads withpolygonal surface.

The dimensions of the spacers dictate the space between the base surfaceand the cover (top surface). Without being limited thereto, thedimensions of the spacers are such that a space of 50 μm, at times 100μm and further at times 200 μm between the two surfaces are formed. Thisis obtained by using spacers with a cross sectional diameter along theirsmaller axis in the range of 50 μm, at times 100 μm and further at times200 μm. Optionally, the spacers have a diameter that is comparable tothe height of the cells in the sample. Thus, they may prevent a coverfrom pressing the cells to an extent that is damaging. Accordingly, thediameter of the spacers may be 2-3 μM. When using spacers in the form ofbeads, the space may be dictated by the beads radius, being, forexample, in the above ranges.

The spacers may be made of any biocompatible material. For example andwithout being limited thereto, latex, silicon, glass, plastic etc.Further, the spacers may be transparent, semi transparent ornon-transparent. They may also be of a type only visible under a givenwavelength or band of wavelengths.

The number of spacers used may be chosen so that it ensures the top andbase surfaces of the carrier to be substantially parallel. To this end,it is sometimes preferable that the spacer units used are of essentiallyuniform size. To maintain a fixed height between the surfaces, thespacers may be fixed to at least one of the surfaces, e.g. the basesurface. This may also reduce the number of spacers needed.

In some embodiments, an amount of spacers (e.g. beards) in a range of 1spacer per mm² to 2500 spacers per mm² is used.

In some embodiments, the space between the base surface and top covermay be dictated by the use of a spring affixed to at least one of thesurfaces.

Spacers may be used in the process of constructing a carrier with anygiven vertical height (e.g. equal to the vertical depth of the fluidsample h). For example, spacers may be mixed in with glue that binds thetop and bottom surfaces of the chamber (mixing beads in glue as used forexample in liquid crystal displays (LCD)).

The present disclosure also provides a kit, the kit comprising:

-   -   a carrier comprising a base surface and a vertical height (H);        and instructions for performing the steps of:        -   providing a cell suspension from a blood sample comprising            red blood cells, the cell suspension being of a cell            concentration (C) determined by the function:            C=F/(h*pi/4*d ²).    -   (F) being a desired base surface coverage; and (d) being an        average cell dimension of the cells in the cell suspension;        -   introducing the cell suspension of the desired concentration            C onto the base surface of the carrier, the cell suspension            having said vertical depth (h) when in said carrier, said            vertical depth (h) being smaller or equal to the vertical            height (H);        -   allowing the cells in the cell suspension to settle on said            base surface of the carrier to form onto the base surface a            monolayer of cells;        -   acquiring at least one microscope image of at least a            portion of    -   wherein said at least one microscope image is obtained by        setting the microscope's magnification to a Depth Of Field (DOF)        that is not more than 20% of the vertical height of the cell        suspension when settled on said base surface.

The kit and the instruction therein allow performing the method asdisclosed herein.

Also provided by the present disclosure is a system for imaging a bloodsample, the system comprising:

-   -   One or more reservoir units for holding, respectively, one or        more sample treatment agents comprising at least one blood cells        diluting agent;    -   a blood sample preparing unit being in fluid communication with        said one or more reservoir units and configured to receive a        blood sample comprising red blood cells and amount of at least        one blood cell diluting agent and to form therefrom a blood        cells suspension, the amount of said at least one cell diluting        agent being determined so as to dilute said sample of cells by a        dilution factor (D) so as to provide a cell concentration (C);    -   a microscope image acquisition unit for acquiring at least one        image of the blood cells suspension when on a base surface of a        carrier, the carrier having a vertical height (H) being greater        or equal to a vertical depth (h) of said cell suspension when on        said base surface;    -   a control unit being configured to:    -   provide dilution factor D of diluting said sample, factor D        being a function of the desired base surface coverage (F), the        average cell dimension d of cell blood cells, and the vertical        depth h of said suspension of cells that provides a monolayer of        the cells when settled on said base surface of the carrier; and    -   acquire at least one microscope image of the cell suspension by        a microscope set to a Depth Of Field (DOF) that is not more than        20% of the vertical height of the cells suspension when settled        on said base surface.

In some embodiment, the control unit is configured to determine thedilution factor based on parameters either being accessed or introducedor already stored within the system. To this end, the control unit isconfigured to access such parameters, including those indicative of adesired base surface coverage.

The system may also comprise an analysis chamber for holding a carrierhaving a vertical height (H) being greater or equal to said verticaldepth (h), said analysis chamber being in fluid communication with saidsample preparing unit and configured to receive into said carrier anamount of cells having a concentration C.

When referring to fluid communication it is to be understood asincluding a conduit, e.g. a pipe, a tube connecting one unit to another,as well as any fluid transfer means such as robotic fluid withdrawal anddischarge devices or pipettes.

As part of the system disclosed herein, the said one or more treatmentagents may comprise, in accordance with some embodiments, one or morestains. At least one of the at least one diluting agent comprises theone or more stains. In other words, the at least one stain may be mixedwith the dilution buffer prior to diluting the blood sample.

In some embodiments, at least one of the stains is a fluorescent stain.In some embodiments, the at least one stain is selected from the groupconsisting of acridine orange, Hoechst family, molecular beacon, Sytoxor DAPI.

In addition and in accordance with some embodiments, the systemcomprises a fluid withdrawal mechanism for withdrawing a sample of bloodcells from a blood source and introduce said sample of blood cells intosaid sample preparing unit.

Further, in some embodiments, the system comprises a cell count unit forcounting cells received by the cell preparing unit.

In yet some further embodiments, the system comprises a sample preparingunit and the latter may comprise an agitating unit configured touniformly distribute the cells in the cell suspension.

As appreciated, the system may also comprise, in accordance with someembodiments, an output port for outputting said at least one image ordata corresponding to the image, for example to an associated displayand/or to a remote location.

Yet further, in accordance with some embodiments, the system maycomprise a memory unit accessible by the control unit and comprisingdata indicative of said input parameters. The data may comprises for oneor more input parameters a cell type in the blood sample and said datais accessible by said control unit. The data may also comprise said forone or more input parameters a cell type in the blood sample and saiddata is accessible by said control unit.

Further, at times, the system may comprise a processor configured toanalyze said at least one microscope image to determine based thereonpresence or absence of a pathogen in said cell suspension.

Preferably, the system is for detecting one or more parasites within ablood sample.

The system also comprises a control unit. In accordance with someembodiments, the controller is configured to cause the microscope imageacquisition unit to acquire a plurality of microscope images of themonolayer, at least two of which are provided under differentconditions, said conditions comprising imaging different portions of themonolayer (or base surface) and different illumination conditions.

Reference is now made to FIG. 1A which is a schematic illustration ofcomponents of a system 100 in accordance with an embodiment of thepresent disclosure.

System 100 includes a reagent reservoir unit 102 for holding a sampletreatment agent and a sample preparing unit 104, the reagent reservoirunit 102 comprises a diluting agent and is in fluid communication withthe sample preparing unit 104 through conduit line 106.

While system 100 is illustrated as including a single reagent reservoirunit 102, as shown in FIG. 1B, the system may similarly be configured toinclude a plurality of reagent reservoir units 104A, 104B, 104C, eachfor holding the same or different agent and being in fluid communicationwith the sample preparing unit 104 via an array of conduits 106A, 106Band 106C. Each reagent reservoir units 104A, 104B, 104C may include thesame diluting agent, in a different concentration, a different dilutingagent, or different treatment agents, such as a stain, a dye, a spaceragent, as further discussed below.

Sample preparing unit 102 is configured to receive a sample of cellsand, if required, from reagent reservoir unit 104, an amount of celldiluting agent. In operation, the biological sample of cells in thesample preparing unit 102 is preferably is in the form of a cellsuspension. To this end, the sample preparing unit may be equipped witha mixing mechanism, such as a gentle stirrer or shaking/agitationplatform etc (not illustrated), so as to cause the cells to suspend inthe medium they are in (i.e. prevent settling of the cells in the samplepreparing unit).

System 100 also comprises an analysis chamber 108 for holding a carrier200 illustrated in FIG. 1C as a well with having a base 202 side walls204, an open top 206, and a vertical height (H).

The analysis chamber 108 in FIGS. 1A and 1B is in fluid communicationwith the sample preparing unit 104 via conduit line 110 and isconfigured, upon operation and when holding a carrier 200 in place, toreceive into carrier 200 an amount of cells, from sample preparing unit104, the amount of cells being delivered at the desired concentration C.Notably, while System 100 is illustrated as including fluidcommunication lined, transfer of matter from one unit to the other mayalternatively or additionally be accomplished manually, e.g. usingtransfer equipment such as a pipette and/or automatically, for exampleby a robotic arrangement.

System 100 also comprises a control unit 112 configured to receive,inter alia, input parameters regarding concentration of cells beforedilution (C₀), an average dimensions (d) for said cells, and desiredbase surface coverage (F). This input may be introduced into controlunit 112 manually, e.g. by a user interface (not illustrated), and/or byretrieval from a stored database. Control unit 112 is also configured toapply Factor D of dilution of the reservoir sample of cells by adiluting agent. Further, control unit 112 is configured to controloperation of the components of the system, including delivery of adetermined amount of diluting agent from a reagent reservoir unit 104into the sample preparing unit 102, delivery of an amount of cells fromthe sample preparing unit 202 into carrier 200 when held in analysischamber 108.

The control unit may also be equipped with a memory unit (notillustrated) operable to store one or more of the input parametersoptionally in association with cell types and/or cell sources as well asa desired surface coverage (F) and desired Factor D as well as any otherparameter for which storing is desired by the user.

At times, system 100 may also include a sample reservoir 114 for holdingthe cell source sample in its base (undiluted) concentration. The samplereservoir 114 is in fluid communication with sample preparing unit 102via conduit line 116.

System 100 may also include or be associated with an imager 118 forimaging cells in carrier 200, after the cells are settled in the form ofa monolayer on the base surface 202 of carrier 200; and in suchembodiments, control unit 112 is further configured to actuate imager118 to acquire at least one image of the cells in carrier 200.

Imager 300 may be any image acquisition device known in the art. In someembodiments, imager 118 is a microscopic camera (e.g. CCD).

Imaging may require, at times, dying of the sample prior to imaging. Tothis end, and as also mentioned above, in system 100 one or more of thereagent reservoir unit 102A, 102B or 102C etc. may include a stainingreagent. At times, the stain in a reagent reservoir may be in fluidcommunication directly with analysis chamber 108 (not illustrated).

Further, at times, in system 100 one or more of the reagent reservoirunits 102A, 102B or 102C etc may include a spacer reagent including forexample, microparticles or beads as discussed above in a suitablebuffer.

System 200 may also comprise or be associated with a cell counting unit220 for counting the number of cells in the sample reservoir (i.e. toestimate the concentration of cells before dilution (C₀) and/or numberof cells in the sample preparation unit (for the desired concentrationC).

System 100 also includes fluid withdrawal mechanisms, such as pumps, andinjectors (not illustrated) configured to withdraw fluid from thedifferent reservoir units and inject the withdrawn fluid into samplepreparation unit 104 and/or carrier 200 when in analysis chamber 108,and from sample preparation unit 104 into analysis chamber 108 asdictated by control unit 112.

System 100 may also comprise input and output utilities 122 and 124,respectively, introducing data into control unit 112 and for presentingat least one image or data corresponding to the at least one imageobtained by the imager.

DESCRIPTION OF NON-LIMITING EXAMPLES Example 1 Detection of Trypanosomabrucei

A mammalian blood sample (human) was assayed as follows for the presenceof Trypanosoma brucei. Typically, such blood samples hold between3,000,000 and 6,000,000 RBC's in each 1 μl (C₀). A cartridge having onemain chamber was manufactured, whereby the height (H) of the chamber was100 μM and the chamber could receive a volume of 1 μl.

The blood sample was diluted 50× (D) to have a surface coverage (F) ofbetween 0.6-0.8 with a 1000 μl solution, containing 1% TRIS-EDTA 1 mM,15 μl Hoechst 1 μg/μl, 2 μl acridine orange 1 μg/μl, 99% bufferedsaline. The sample was loaded into the chamber and the chamber wastransferred to a microscope stage for imaging in both brightfield and influorescence at excitation 370 nm and 475 nm and emissions ofapproximately 433 nm and 517 nm, and 613 nm using an automatedmicroscopy device. Sedimentation of the sample occurred at about 1second per 1 μM height of the chamber (H), which is approximately 90seconds.

Microscope images were acquired 1-2 minutes after introducing the bloodinto the chamber. The images were taken at a 20× magnification, with adepth of field (DOF) of about 2.3 μm for the florescent image (i.e.about 2.3% of the sample height).

As seen in a brightfield image shown in FIG. 2, the cells formed amonolayer where some cells were touching and almost no cells wereoverlapping. As seen, the sample included predominantly red blood cellswhich covered about 70% of the surface (F≈0.7). In addition, Trypanosomabrucei were observed and marked by arrows. FIG. 3 depicts the same cellsimaged fluorescently (excitation at 350 nm and emission measured at 461nm) to specifically highlight Trypanosoma brucei against the backgroundof red blood cells and allows their detection.

Example 2 Detection of Plasmodium falciparum

Staining Solution Composition

The purpose of this example stain solution is to identify live pathogen(e.g. Plasmodium) inside living blood cells. The solution comprisesHoechst 33342 (excitation 350 nm) and Acridine Orange (excitation 500nm). The dyes were mixed with saline and Tris-EDTA to create an isotonicsolution to keep red blood cells at physiological conditions during thestain and prevent them from lysing. This solution was used as a dilutionsolution thus potentially providing dyes and diluting the cells in asingle step.

Stating Blood Sample for Detection of Plasmodium

Blood previously mixed with EDTA (or any other anti-coagulant) wasdiluted in the above stain solution (˜1:100). Within 10 seconds theblood was stained with the chemical dyes and giving off fluorescentsignals between 450 nm and 550 nm when appropriately illuminated. Thesolution mixed with the blood was loaded into a plastic cartridge. Afterthe blood cells settled they were scanned using LED fluorescent lightsand a fluorescent microscope.

Hoechst was intended to stain DNA while Acridine Orange was meant tostain the RNA in the cells. In normal mature red blood cells there is noDNA or RNA, so mature red blood cells showing a positive stain may beindicative of an intracellular pathogen, such as malaria.

Detection of Trypanosoma brucei

An assay similar to that of Example 1 was performed with a blood samplesuspected to be infected with Plasmodium falciparum. In general, a wholeblood sample was diluted by a factor of 100 (1:100) in a fluorescentstain solution comprising Tris-Saline and the fluorescent dye AcridineOrange. The diluted cell sample/suspension was introduced into a chamberhaving a height (H) of about 200 μm and filled it up, thereby achievinga vertical depth of about 200 μm. The cells were then allowed to settleand form a monolayer.

FIG. 4A shows a brightfield image at 20× magnification of the monolayerof cells, with an apparent surface base coverage of about 75% (F≈0.75).FIG. 4B shows a florescent image of the same cells of the diluted bloodsample stained with the staining solution, showing in this image thestain by Acridine Orange (AO) fluorescent dye, and emitting fluorescenceat 570 nm. The DOF was 2.3 μM (ca. 1.12% of the sample height (h)). TheRBC depicted in this Figure had a maximal dimension of about 4 μM.

Detection of Plasmodium and Platelets in a Blood Sample

A differential detection between Plasmodium and platelets in a bloodsample comprising red blood cells was also performed, using the sameassay conditions and parameters as in the above FIG. 4A-4B.

FIG. 5A is a brightfield image showing the spread, in the form of acontinuous layer, of cells. FIG. 5B is a florescent image (emissions at460 nm) showing the platelets and malaria infection (with two parasitesin the same RBC, thus terms trophozoites). The florescent signal of themalaria parasite is stronger and distinguishable from that exhibitedfrom the platelets, having similar size.

The invention claimed is:
 1. A method comprising: introducing a cellsuspension comprising red blood cells, into a carrier that includes abase surface; allowing the cells in the cell suspension to settle on thebase surface of the carrier for a period of time that is at least 1second per micrometer (μm) of a vertical height of the cell suspensionwhen on the base surface, such as to form a monolayer of cells on thebase surface of the carrier; and acquiring at least one microscope imageof at least a portion of the monolayer of cells, the at least onemicroscope image being obtained by a microscope set to Depth Of Field(DOF) that is not more than 20% of the vertical height of the cellsuspension when settled on the base surface.
 2. The method of claim 1,wherein the vertical height of the cell suspension when settled on thebase surface is between 20 μm and 1000 μm.
 3. The method of claim 2,wherein the at least one microscope image is obtained by a microscopeset to a DOF that is between 0.5 μm and 10 μm.
 4. The method of claim 1,wherein the at least one microscope image is obtained by a microscopeset to a DOF that is not more than 15% of the vertical height of thecell suspension when settled on the base surface.
 5. The method of claim1, further comprising, prior to the introducing, diluting a blood samplecomprising the cells by a dilution factor (D) to obtain the cellsuspension.
 6. The method of claim 5, wherein the dilution factor (D) isbetween 50 and
 300. 7. The method of claim 1, further comprising formingthe cell suspension by diluting a blood sample comprising red bloodcells to obtain a cell concentration such that, after the cellsuspension settles on the base surface, the density of the cells of thecell suspension is between about 10,000 and about 30,000 cells per mm².8. The method of claim 7, wherein forming the cell suspension comprisesforming the cell suspension such that, after the cell suspension settleson the base surface, the cell suspension forms a monolayer having anaverage base surface coverage of between 40 percent and 90 percent. 9.The method of claim 1, further comprising selecting a microscopeobjective lens that provides the DOF, wherein the lens permits acquiringan image of at least one object being no more than 3 μm long at anydimension thereof.
 10. The method of claim 9, wherein selecting themicroscope objective lens comprises selecting a lens that permitsacquiring an image of at least one object having a height with respectto the base surface of no more than 3 μm.
 11. The method of claim 1,further comprising analyzing the at least one image to determine basedthereon presence or absence of a pathogen in the cell suspension. 12.The method of claim 11, wherein analyzing the at least one image todetermine based thereon presence or absence of a pathogen in the cellsuspension comprises analyzing the at least one image to determine basedthereon presence or absence of a blood infecting protozoa in the cellsuspension.
 13. The method of claim 12, wherein the blood infectingprotozoa is selected from the genus consisting of Trypanosoma,Plasmodium; Toxoplasma and Babesia, and wherein analyzing the at leastone image to determine based thereon presence or absence of the pathogenin the cell suspension comprises analyzing the at least one image todetermine based thereon presence or absence of the blood infectingprotozoa from the selected genus in the cell suspension.
 14. The methodof claim 1, wherein allowing the cells in the cell suspension to settleon the base surface of the carrier to form the monolayer of cells on thebase surface of the carrier comprises allowing a period of time of lessthan 5 minutes for the cells to settle as a monolayer on the basesurface.
 15. The method of claim 1, wherein a vertical height of thecarrier is between 20 μm and 300 μm.
 16. The method of claim 1, whereinacquiring the at least one microscope image comprises acquiring aplurality of microscope images, at least two of which are provided underdifferent conditions, the different conditions being selected from thegroup consisting of: different portions of the base surface beingimaged, and different illumination conditions being used.
 17. The methodaccording to claim 1, wherein introducing the cell suspension into thecarrier comprises introducing the cell suspension into the carrier, thecell suspension having a concentration that is such that whensubstantially all of the cells in the cell suspension are allowed tosettle on the base surface of the carrier to form the monolayer ofcells, a monolayer is formed on the surface with substantially nooverlap between the cells.
 18. A method for imaging a blood sample, themethod comprising: introducing a cell suspension, onto a base surface ofa carrier, wherein the cells are human cells comprising at least 75% redblood cells and at least a portion of the cells are suspected of beinginfected with a plasmodium parasite selected from the group consistingof Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax),Plasmodium ovale (P. ovale), Plasmodium malariae (P. malariae), andPlasmodium knowlesi (P.knowlesi); mixing the cells with one or morestains, the one or more stains including at least one fluorescent stain;allowing the cells in the cell suspension to settle on the base surfaceof the carrier, such as to form a monolayer of cells on the base surfaceof the carrier; and acquiring at least one microscope image of at leasta portion of the monolayer of cells, the at least one microscope imagebeing obtained by a microscope set to Depth Of Field (DOF) that is notmore than 20% of a vertical height of the cell suspension when settledon the base surface, the acquiring of the at least one microscope imagecomprising acquiring a fluorescent image that is configured tofacilitate detecting staining with the fluorescent stain.
 19. A systemfor imaging a blood sample, the blood sample including human cells thatinclude at least 75% red blood cells, at least a portion of the cellsbeing suspected of being infected with a plasmodium parasite selectedfrom the group consisting of Plasmodium falciparum (P. falciparum),Plasmodium vivax (P. vivax), Plasmodium ovale (P. ovale), Plasmodiummalariae (P. malariae), and Plasmodium knowlesi (P.knowlesi), the bloodsample including a blood cell diluting agent, the system comprising: oneor more stains configured to be mixed with the blood sample, the one ormore stains comprising at least one fluorescent stain; a blood samplepreparing unit configured: to receive the blood sample, and tofacilitate forming therefrom a blood cell suspension, that is such thatthe cell suspension forms a monolayer of the cells when allowed tosettle on a base surface of a carrier; a microscope image acquisitionunit configured to acquire at least one image of the blood cellsuspension when on the base surface of the carrier, the at least oneimage including at least one fluorescent image that is configured tofacilitate detecting of staining the fluorescent stain; a controllerconfigured to drive the microscope image acquisition unit to acquire theat least one microscope image of the cell suspension using a microscopeset to a Depth Of Field (DOF) that is not more than 20% of a verticalheight of the cell suspension when settled on the base surface.
 20. Thesystem of claim 19, wherein the controller is configured to drive themicroscope image acquisition unit to acquire a plurality of microscopeimages of the monolayer, at least two of which are provided underdifferent conditions, the different conditions being selected from thegroup consisting of: different portions of the monolayer being imaged,and different illumination conditions being used.
 21. The systemaccording to claim 19, wherein the blood sample preparing unit isconfigured to facilitate forming a blood cell suspension, that is suchthat that when substantially all of the cells in the cell suspension areallowed to settle on the base surface of the carrier to form themonolayer of cells, a monolayer is formed on the base surface withsubstantially no overlap between the cells.
 22. A system for imaging ablood sample comprising red blood cells and at least one blood celldiluting agent, the system comprising: a blood sample preparing unitcomprising a carrier having a base surface, the blood sample preparingunit being configured: to receive the blood sample, and to facilitateforming therefrom a blood cell suspension, that is such that the cellsuspension forms a monolayer of the cells when allowed to settle on thebase surface of the carrier, by allowing the cells in the cellsuspension to settle on the base surface of the carrier for a period oftime that is at least 1 second per micrometer (μm) of a vertical heightof the cell suspension when on the base surface; a microscope imageacquisition unit configured to acquire at least one image of the bloodcell suspension when on the base surface of the carrier; a controllerbeing configured to drive the microscope image acquisition unit toacquire at least one microscope image of the cell suspension by amicroscope set to a Depth Of Field (DOF) that is not more than 20% of avertical height of the cell suspension when settled on the base surface.